Our clinical studies have established that impaired alveolar epithelial fluid clearance is associated with a higher mortality in patients with acute lung injury. New studies are needed to understand the biological and clinical mechanisms that impair alveolar epithelial fluid clearance in patients with acute lung injury. It is also important to test therapeutic strategies that may increase the clearance of alveolar edema fluid in the human lung. Therefore, in Aim 1, we will use pulmonary edema fluid and plasma collected from critically ill patients with acute lung injury to study the mechanisms that mediate an increase in protein permeability and a decrease in vectorial fluid transport across cultured human alveolar epithelial type II cells that form tight monolayers in transwell filters grown in an air-liquid interface. Control studies will be done with pulmonary edema fluid and plasma from patients with hydrostatic, cardiogenic pulmonary edema. In Aim 2, we will collect sequential samples of pulmonary edema fluid and plasma from patients with early acute lung injury to (1) measure alveolar fluid clearance, (2) biological markers of lung epithelial and endothelial injury, and (3) clinical measurements of lung function including the pulmonary dead space fraction, respiratory compliance and oxygenation in order to assess the combined predictive value of both biologic and physiologic mechanisms that contribute to impaired alveolar fluid clearance in clinical lung injury. In Aim 3, we will use a novel preparation of ventilated and perfused human lungs harvested from brain dead donors to measure both lung endothelial and epithelial permeability and alveolar epithelial fluid clearance to test the effect of several clinically relevant therapeutic strategies (low tidal volume, high frequency ventilation, beta-2 adrenergic agonist, dopamine, interleukin-1 receptor antagonist, and inhibitors of transforming growth factor- b) on lung fluid balance over 4-8 hours. In summary, the integrated studies of biochemical, physiological and clinical measurements in critically ill patients in aim 2 will generate new insights into the mechanisms that impair alveolar epithelial fluid clearance in clinical acute lung injury. The proposed studies in aims 1 and 3 studies will provide innovative, novel approaches that use human alveolar type II cells and human lungs to study the mechanisms of alveolar epithelial injury as well as to test the potential value of several clinically relevant therapeutic strategies.